This study evaluated the antimicrobial activity of root canal filling pastes commonly used in primary teeth: iodoform paste, zinc oxide-eugenol (ZOE), and analytical grade calcium hydroxide (Ca[OH]2). The materials were evaluated in the following 6 combinations: iodoform paste (Rifocort [prednisolone + rifampicin] + iodoform + camphorated paramonochlorophenol); zinc oxide + eugenol in a proportion of 1:3; zinc oxide + eugenol in a proportion of 1:5; Ca(OH)2 + water; iodoform paste + ZOE (1:3) + Ca(OH)2; and ZOE (1:3) + Ca(OH)2. Chlorhexidine gel (0.2%) served as the positive control. A commercial strain of Enterococcus faecalis (ATCC 25175) was plated in petri dishes containing 10 mL of brain-heart infusion agar culture medium with a hole in the center. The filling pastes (3 g) were manipulated and inserted in the hole. The plates were incubated at 37°C for 48 hours, and then the inhibition zone associated with each paste was measured. The experiments were carried out in triplicate (n = 3 per group). The data were submitted to analysis of variance and Tukey tests (P 0.05). Of the test materials, the combination of iodoform paste + ZOE + Ca(OH)2 presented the second largest inhibition zone, 6.27 (3.08) mm, significantly different from that of the non-iodoform pastes (P < 0.05). It can be concluded that iodoform paste, alone or in combination with ZOE + Ca(OH)2, demonstrates high antimicrobial activity, while the combination of ZOE + Ca(OH)2 demonstrates moderate activity.